Experiment 2
Determination of Vitamin C
content in vegetables and fruits
Preface: Physiological Functions

Vitamin C, namely ascorbic acid, is vital for us:
– Electron donor with powerful antioxidant capacity



Protect and even re-generate other antioxidants: Vit.E, Vit.A, GSH
Facilitate gastrointestinal absorption of iron
Inhibit the synthesis of N-nitrosamine from nitrite and nitrate
– Play a role in hydroxylation reactions





Detoxification
Collagen synthesis: triple helix is formed after hydroxylation of proline
and lysine (proline/prolyl hydroxylase; lysine/lysyl hydroxylase)
Biosynthesis of neurotransmitters: norepinephrine from dopamine
(dopamine beta hydroxylase); 5-hydroxi-tryptophan from tryptophan
Synthesis of carnitine essential for the transport of fatty acids into
mitochondria for ATP generation
Promote cholesterol metabolism and excretion
– Prevent scurvy: most important function
Preface: Properties

Easily soluble in water, so easily lose by over-washing or
with discarded cooking soup

Easily destroyed by oxidation:
– Heat or prolonged cooking
– Exposure to air or alkaline medium
– Contact with copper or iron
 Determination of vitamin C
• Determination of total Vitamin C
• Determination of reduced Vitamin C
Reduced
De-hydrogen
Part 1
Determination of reduced
Vitamin C content
(by 2,6-dichlorophenol indophenol)
Principle



DCPIP (2,6-dichlorophenol indophenol) is a kind of red dye in
possession of oxidizing capacity
DCPIP loses red when reduced by reduced ascorbic acid through
redox reaction
– DCPIP fades when reduced ascorbic acid is excessive
– DCPIP displays its original red color once the reduced ascorbic
acid is depleted
Therefore, the content of reduced ascorbic acid can be calculated
according to the consumed DCPIP during titration
– End point: light red (over 15 seconds)
Amount of
consumed dye
direct
proportion
Reduced ascorbic
acid content
Procedure

Demarcation of DCPIP solution

Determination of sample

Calculation
Determining the content of DCPIP
Standard
ascorbic acid
solution
Titrated by
dye
Titrated by
0.001M KIO3
Concentration
of ascorbic
acid solution
Concentration of
dye solution
The result is:
1ml dye solution
T=0.088
0.088mg ascorbic acid
1.2.2
Determining the reduced forms in sample
sample
100g of sample
cutting, pounding with
100ml 2% oxalic acid
homogenate
pentanol
weighing 10g 50ml cylinder washing beaker Diluting to
with beaker
with a lid 1% oxalic acid graduation
Shaking well
20ml upper
clear solution
taper
bottle
white bole color
shaking fade
Keeping still
filtering
20ml 1%
oxalic acid
blank
taper white bole Keeping still
bottle shaking
5ml filtrate
5ml filtrate
titrating
light red, keeping 15sec
1.2.3 calculation
T=0.06
The content of reduced ascorbic acid (mg/100g) 
TTT=0.062mgVC/1ml dye
(V1V0)T
100
W
2. Determination of total ascorbic acid
content by 2,4-dinitrophenyl hydrazine
2.1 Principle:
Reduced oxidating De-hydrogen
Vit.C
form
red osazone
2,4-dinitrophenyl
hydrazine
dissolving with
making colorimetric assay
85%H2SO4
There is a direct proportion
between the amount of osazone
and total ascorbic acid.
total Vit. C
2.2 procedure

2.2.1 Sample extracting:
4g homogenate diluting ,washing
with 1% oxalic acid
(beaker)
volumetric
flask (100ml)
1% oxalic acid
diluting to graduation
filtering
2.2.2 oxidation
25ml filtrate
100ml taper 0.5g carbon active
shaking 30sec
bottle
filtering
10ml oxidated filtrate
10ml 1% oxalic acid solution
mixing well
2.2.3 Formation of osazone

Getting three tubes and process as follow.
number
A
B
C
Oxidation filtrate(ml)
10% thioura(drop)
2.0
1
2.0
1
2.0
1
0
0.5
0.5
2,4-dinitrophenyl hydrazine(ml)
 A:
blank tube.
B,C: sample tube.
Keeping these tubes at 37oC in water bather for 3 hours,
take them out and keep at room temperature, add 0.5ml
2,4-dinitrophenyl hydrazine into tube A.

2.2.4 Dissolving of sha

each tube
adding 2.5ml 85% H2SO4 slowly along
the tube wall, cold water bathe, shaking
taking out
keeping still for 30min
490nm adjusting zero with tube A
colorimetric analysis
2.2.5 Formation of standard curve
standard ascorbic acid applying
solution(100ug/ml) about 25ml
100ml taper bottle
adding 0.5g carbon active, shaking 1 min
filtering
10ml filtrate
diluting with 1% oxalic acid
100ml volumetric flask
final conc.: 10ug/ml

Get five tubes process as follows:
number
0
Standard ascorbic acid solution (10ug/ml)(ml) 0
Content of ascorbic acid in each tube(ug)
0
1 2
3
4
0.5 1.0 1.5 2.0
5
10 15 20
1% oxalic acid (ml)
10% thioura (drop)
2.0 1.5 1.0 0.5 0
1
1
1
1
1
2%2,4-dinitrophenyl hydrazine
0.5 0.5 0.5
0.5
Doing as the same as the above described for the sample.
Drawing standard curve with the light density as ordinate, and
the content of ascorbic acid as abscissa.
0.5
Light density
standard curve
0
calculation
5
10
15
20
content of ascorbic acid(ug)
C
1
content of total ascorbic acid in sample(mg/ 100g)  
100
W 1000
C:ug of standard ascorbic acid correspond to sample tube.
W:g of sample in sample determination liquid
Questions:
1.
Why do we always use oxalic acid in the
whole process?
2.
The concentration of oxalic acid on making
homogenate is 2%, but in other steps, it is 1%.
The concentration is different ,why?